Purification of monoclonal antibodies

ABSTRACT

A method of chromatographically separating monoclonal antibody type IgM from mouse ascites fluid utilizing a particular chromatographic packing of silica gel bearing bound polyethylenimine functions.

The invention relates to a method of purifying the monoclonal antibodycontent of a mouse ascites fluid sample. More particularly, the presentinvention is concerned with a method of separating and purifyingmonoclonal antibody type IgM from mouse ascites fluid utilizing liquidcolumn chromatography on a particular stationary porous phase of silicagel bearing bound polyethylenimine (PEI) functions and gradient elutionof the monoclonal antibody from the polyethylenimine bound column withaqueous buffer of from about pH 6.0 to about pH 8.3.

The particular stationary porous phase of silica gel bearing boundpolyethylenimine functions, that is, the chromatographic packingutilized in this invention, are of two types.

The preferred type is described in U.S. patent application Ser. No.555,368, now U.S. Pat. No. 4,540,486 filed by Hugh Ramsden on Nov. 25,1983, and entitled "Polyethylenimine Bound Chromatographic Packing", thecontent of which is incorporated herein by reference. Relevant text ofthis application is reproduced below.

The second type is the adsorbed cross-linked PEI-silica gel stationaryphase described by G. Vanecek & F. E. Regnier, Anal. Biochem. 121,156-159 (1982) and A. J. Alpert & F. E. Regnier, J. Chromatogr. 185,375-392 (1978), which type is commercially available from SynChrom, Inc.of Linden, Indiana, under the brand name "SynChropak". Alpert andRegnier have shown that polyethyleneimine (PEI) may be adsorbed tosilica gel surfaces and crosslinked to form a stable polyamine layer.The structure of PEI provides sufficient primary and secondary aminogroups that adjacent adsorbed PEI molecules on the surface of silica gelmay be crosslinked by multifunctional oxiranes into a polymeric layer.Through the use of a hydrophilic crosslinker such as diglycidylethyleneglycol, a hydrophilic coating may be produced.

DETAILED DESCRIPTION OF (RAMSDEN) INVENTION

The non-crosslinked covalently bound PEI silica gel products of thepresent invention are conveniently prepared in accordance with thefollowing steps:

A. reacting particulate silica gel having an average particle diameterof from about 3 to about 70 microns and an average pore size of fromabout 50 to about 1000 Angstrom units in an inert organic solvent slurrywith a lower alkanolic solution of polyethyleniminopropyl trimethoxysilane having an average molecular weight of from about 400 to about1800, said reaction being conducted at ambient to refluxing temperaturefor about 2 to about 50 hours;

B. recovering the resultant solid fraction from the reaction mixture;and

C. heating said solid fraction at a temperature and for a timesufficient to dry and completely bond the silane to the silica gel.

As used herein, the term "covalently bound" or "covalently bonded" meansthat the PEI moieties are covalently attached to the silica gel by wayof chemical interaction resulting in a propyl-silyl (PrSi) linkage; andthe term "non-crosslinked" means that the imino and amino groups onadjacent covalently bound PEI moieties are not crosslinked, or reactedwith a crosslinking agent, to form a polymeric layer.

Without being bound thereby, it is believed that the reaction proceedsto completion in two steps as follows:

Step 1: Silica hydroxyls and the methoxy groups on the silane react toform Si--O--Si bonds and free methanol, with some residual methoxygroups remaining unreacted: ##STR1## Step 2: Completion of the reactionwith the residual methoxy groups is effected during heat curing by (a)and (b): ##STR2##

Silica gel, consisting of amorphous silica, is commercially available inirregular and spherical (preferred) particulate forms and in severalcommercial grades with mesh sizes ranging from 3 through 325 (ASTM).Rather than relying upon a numerical indication of mesh size, however,more accurate indicia for purposes of this invention are the averagediameter and average pore size of the silica gel particles,respectively, from about 3 to about 70 microns and from about 50 toabout 1000, preferably 250-500, Angstrom units. For end product use inpacking HPLC chromatographic columns, a silica gel starting material offrom about 3 to about 10 microns is preferred, and, for packing lowpressure chromatographic columns, from about 40 to about 70 microns ispreferred.

Among the inert organic solvents suitable for preparing the silica gelslurry are aliphatic hydrocarbons such as, for example, hexane, heptaneand the like; aromatic hydrocarbons such as, for example, benzene,toluene, xylene and the like; lower alkanols such as, for example,ethanol, isopropanol, butanol and the like; chlorinated methanes suchas, for example, methylene chloride, chloroform, carbon tetrachlorideand the like (Caution: such chloro solvents may react at highertemperatures-); and such other inert solvents as tetrahydrofuran, glyme,diglyme and the like. In general a 1:5 ratio of silica gel in grams tosolvent in milliliters affords a suitable slurry. Due to the fine,insoluble nature of the particulate silica gel a slurry rather than atrue solution is obtained.

Polyethyleniminopropyl trimethoxy silane, also known as(N-trimethoxysilylpropyl)-polyethylenimine, is the reaction product ofpolyethylenimine and aminopropyltrimethoxy silane and can be representedby the following formula: ##STR3## wherein, for purposes of thisinvention, n is an integer from about 4 to about 37, or, if expressed interms of average molecular weight, from about 400 to about 1800.

The silane (I) is used in the reaction with the silica gel in the formof a lower C₁ -C₆ alkanolic solution using sufficient alkanol tosolubilize the silane. A fifty percent w/w isopropanolic solution ispreferred. In general, about 25-100 grams of the silane, or,alternatively, about 50-200 ml of a fifty percent w/w alkanolic solutionof the silane, is used to react with each 100 grams silica gel. Thereaction may be conducted at ambient temperature although elevatedtemperatures up to the refluxing temperature of the reaction solventsystem may be utilized to enhance the rate of reaction. The reactionproceeds readily to substantial completion (Step 1) within 2-50 hours.Stirring during admixture of the reactants is advantageously employedalthough the reaction thereafter may continue without further stirring.Anhydrous conditions are not critical, it having been found that thepresence of a small amount of water, for example, about 0.1-1.0 ml per50 ml of the slurry solvent, does not adversely affect the reaction.

The resultant solid fraction is recovered from the reaction mixture byconventional physical means, for example, filtration, centrifugation,etc. In general, a filtering means sufficient to retain a particle sizeof 5 microns is suitable whereas centrifuging is suitable for a particlesize of 3 microns.

The recovered solid fraction is then heat cured at a temperature and fora time sufficient to dry and completely bond the silane to the silicagel covalently. In general, from about 1-4 hours at about 40°-120° C.has been found sufficient. The thus-obtained covalently bound,non-crosslinked final product preferably contains from about 0.5 toabout 3.8 percent nitrogen.

For purposes of this invention, the chromatographic packing, hereinaftersometimes referred to as "PEI-silica gel" for purposes of convenience,is utilized wherein the starting silica gel is limited to one having anaverage particle diameter of from about 3 to about 40 microns. Theaverage pore size may be from about 50 to about 1000 Angstrom units,although an average pore size greater than 250 Angstroms is preferred.

Accordingly, the instant invention provides a method of obtainingessentially homogeneous monoclonal antibody type IgM from a sample ofmouse ascites fluid containing said monoclonal antibody by employingliquid chromatographic means wherein the chromatographic packingcomprises particulate silica gel having an average particle diameter offrom about 3 to about 40 microns and an average pore size of from about50 to about 1000 Angstrom units to which polyethylenimine functions arebound, either in adsorbed crosslinked form according to Regnier et al.,ibid, or in covalently bound non-crosslinked form according to Ramsden,ibid. Regarding the latter, the PEI-silica gel chromatographic packingis the reaction product of the aforementioned particulate silica gelwith polyethyleniminopropyl trimethoxy silane having an averagemolecular weight of from about 400 to about 1800.

It has now been found that such chromatographic packing is particularlysuitable for use in liquid chromatography, particularly high performanceliquid chromatography (HPLC), for binding type IgM monoclonal antibodiesand other proteins in mouse ascites fluid containing same, therebyallowing preferential separation of the bound proteins by gradientelution using appropriate aqueous buffers. There are thus obtained anumber of protein components which are originally present in the ascitesfluid including the essentially homogeneous monoclonal antibody ofinterest. As used herein "essentially homogeneous" means that >90% ofthe protein present in the quantitatively recovered IgM antibody eluantfraction is the IgM monoclonal antibody. The percent purity may beverified by known procedures in the art, such as, for example, by sodiumdodecyl sulfate polyacrylamide gel electrophoresis, see U. K. Laemmli,Nature, 227, 680 (1970).

The instant invention is suitable for use with mouse ascites fluidcontaining monoclonal antibody of the IgM type. The methodology ofpreparing such mouse ascites fluid containing monoclonal antibody iscommon to the art, for example, see "Monoclonal Antibodies, Hybridomas;A New Dimension in Biological Analyses", by R. H. Kennet et al.,published by Plenum Press, N.Y. 1980; and "Methods in Enzymology" by G.Galfred and C. Milstein, edited by J. J. Langone and H. Van Vunakis,Vol. 73, p. 31-46, publ. by Academic Press, 1981.

The isolation of monoclonal antibody in highly purified form isobviously desirable. For example, for in vivo therapeutic purposes,monoclonal antibody as pure and as concentrated as possible is requiredto minimize adverse side effects and to maximize the intendedtherapeutic purpose. Similarly, for in vitro diagnostic purposes, suchpurified and concentrated monoclonal antibody is desirable to maximizethe sensitivity and specificity of the particular diagnostic test.

Before the mouse ascites fluid can be used for this invention, it ispre-treated to remove interfering particulate matter and is equilibratedto the appropriate ionic strength and pH necessary to achieve binding ofthe monoclonal antibody to the PEI-silica gel support. The particulatematter can be removed by conventional clarifying means, for example, byfiltration or by centrifugation at a force sufficient to pelletize theparticulate material. Equilibration of the particulate-free mouseascites fluid can be achieved by any means common to thestate-of-the-art, for example, by chromatographic desalting with anappropriate buffer on molecular sieves of appropriate type and pore sizesuch as those commercially available under the brand name "Sephadex", bydialysis against an appropriate buffer, and the like, to equilibrate themouse ascites fluid to a pH greater than the pI (that pH at which themonoclonal antibody carries no net ionic charge in its environment) ofthe IgM monoclonal antibody and to an ionic strength equal to or lessthan the ionic strength of the lower ionic strength buffer used forgradient elution in the subsequent chromatographic treatment of themouse ascites fluid.

Chromatographic columns suitable for use in liquid chromatography,preferably HPLC, are packed with the previously described porousPEI-silica gel solid support. Suitable steel or glass chromatographiccolumns include those having dimensions of about 5-100 cm in length andinternal diameters of about 1-100 mm. Selection of the properchromatographic parameters such as, for example, column packingtechnique, column size, column temperature, pressure and the like, arereadily determined by one of ordinary skill in the art.

The packed column is equilibrated in a chromatograph by passing anappropriate buffer solution through the column. After thisbuffer-equilibration step, the column is used to make thechromatographic separation of the proteinaceous components of the mouseascites fluid which, as noted, previously has been freed of particulatematter and has been equilibrated to the appropriate ionic strength andpH. A sample of such pre-treated mouse ascites fluid is then applied tothe buffer-equilibrated column to bind its component proteins to thePEI-silica gel packing.

The bound proteins can then be selectively eluted by conventionalgradient elution techniques, taking into consideration theinterdependent chromatographic parameters of time, flow-rate andgradient shape to generate gradients of increasing ionic strength ordecreasing pH. Anionic buffers, for example, potassium phosphate,tris-acetate, ammonium acetate and the like, of from about pH 6.0 toabout 8.3, can be used to generate such gradients to elute the boundproteins from the polyethyleneimine function. For example, the gradientcan be advantageously formed from about one-half hour to about fourhours with a flow rate of from about 0.1 mL/min to about 2 L/min.

The resolved proteins can be identified by any means common to thestate-of-the-art, for example, by monitoring the ultraviolet absorbanceat 280 nm. The eluent fractions containing the separated proteins can becollected by use of a fraction collector. The eluent fraction containingthe homogeneous monoclonal antibody can be identified by meanswell-estabilshed in the art such as, for example, by a radioimmunoassaydeveloped for the particular antibody, by other antibody-antigenreactions, or by polyacrylamide gel electrophoresis.

The process of this invention has been found to be independent of thetotal volume of the mouse ascites fluid containing the monoclonalantibody and there is no limiting factor except for the amount ofPEI-silica gel used as the chromatographic packing, that is, the processis operable so long as the capacity of the solid chromatographic supportis not surpassed.

In accordance with the method of the present invention, therefore, asample of mouse ascites fluid containing monoclonal antibody type IgM ischromatographically separated to provide said antibody in essentiallyhomogeneous form. As more fully described heretofore, the preferredmethod comprises purifying a sample of particulate-free mouse ascitesfluid containing such monoclonal antibody by:

a. equilibrating said sample of particulate-free mouse ascites fluid toan ionic strength equal to or less than the ionic strength of the lowerionic strength buffer, used for gradient elution in the subsequentchromatographic separation and recovery step and to a pH greater thanthe pI of the IgM monoclonal antibody; and

b. separating and recovering said monoclonal antibody type IgM from saidsample by employing liquid chromatographic means wherein thechromatographic packing consists essentially of the reaction product ofparticulate silica gel having an average particle diameter of from about3 to about 40 microns and an average pore size of from about 50 to about1000 Angstrom units with polyethyleniminopropyl trimethoxy silane havingan average molecular weight of from about 400 to about 1800.

The invention will be more easily understood with the aid of theexamples which follow below which are given solely as an illustration ofthe present invention and are not limitative of the same.

EXAMPLE 1

A slurry of 20 grams silica gel with average particle diameter of 5.25microns and average pore size of 330 Angstroms, commercially availablefrom The Sep A Ra Tions Group, Hesperia, Calif., as a spherical silicaunder the trademark "Vydac A", Catalog No. 101T9B5, in 100 ml tolueneand 2 ml water is prepared and stirred for 10 minutes at roomtemperature. To this is added with stirring 39.4 grams of a 50% w/wisopropanolic solution of polyethyleniminopropyl trimethoxy silanehaving an average molecular weight of 500 and the mixture is stirred foran additional 5 minutes. The mixture is then allowed to stand overnightat room temperature. The mixture is next filtered using a 1.0 micronfilter funnel. The filtrate is washed with 50 ml toluene twice and 50 mlmethanol twice, then air dried on the funnel and finally oven dried at80°-85° C. for about 3 hr. 30 min. to yield the covalently bound,non-cross-linked PEI-silica gel product; about 2.85% N.

EXAMPLE 2

A 2 mL sample of mouse ascites fluid containing a monoclonal antibodybelonging to the class IgM immunoglobulins, as determined by specificantisera, was centrifuged at 15,600 x gravity for 5 minutes. Thesupernatant fluid was then equilibrated to 10 mM potassium phosphatebuffer, pH 6.73, by dialysis overnight (about 17 hours) against 21-liter changes of the 10 mM potassium phosphate buffer, pH 6.73. Astainless steel chromatographic column, 25 cm×0.46 mm, was packed with3.8 g of the PEI-silica gel product obtained from Example 1 andequilibrated with 0.01M potassium phosphate buffer, pH 6.73, by pumpingthis buffer through the column at a flow rate of 1 mL/min for 20minutes. A 0.25 mL equilibrated sample of mouse ascites fluid wasapplied to the polyethyleneimine-bound silica gel column and proteinseparation was achieved by gradient elution using a 60 minute lineargradient from 0.01M potassium phosphate, pH 6.73, to 0.25M potassiumphosphate, pH 6.8, at a flow rate of 1 mL/min. Protein elution wasdetected using UV absorbance at 280 nm with full scale absorbance set at0.64 absorbance units. A series of 280 nm absorbing peaks were detectedranging in retention time from about 3 minutes to about 50 minutes. Theprotein peak at about 44 minutes was identified as the monoclonalantibody by co-chromatographing with a homogeneous sample of the samemonoclonal antibody using the identical gradient profile as outlinedabove.

The protein peak eluting at about 44 minutes was evaluated to be greaterthan 90% pure by two criteria. First, the collected protein peak gave asymmetrical peak after chromatogaphy on the polyethyleneimine-boundsilica gel column, according to F. B. Regnier, Science 222, 245 (1983).Second, sodium dodecyl sulfate polyacrylamide gel electrophoresis,performed essentially as described by U. K. Laemmli, Nature, 227, 680(1970), gave two major coomassie blue bands corresponding to the heavyand light chains of the IgM monoclonal antibody and a third faintcoomassie blue band representing less than 10% of the total stainedprotein.

I claim:
 1. A method of obtaining essentially homongeneous monoclonalantibody type IgM from a sample of mouse ascites fluid containing saidmonoclonal antibody which comprises separating and recovering saidmonoclonal antibody from said sample by employing liquid chromatographicmeans wherein the chromatographic packing consists essentially ofparticulate silica gel having an average particle diameter of from about3 to about 40 microns and an average pore size of from about 50 to about1000 Angstrom units to which polyethylenimine functions are bound incovalently bound non-crosslinked form or in adsorbed crosslinked form.2. A method of obtaining essentially homongeneous monoclonal antibodytype IgM from a sample of mouse ascites fluid containing said monoclonalantibody which comprises separating and recovering said monoclonalantibody from said sample by employing liquid chromatographic meanswherein the chromatographic packing consists essentially of the reactionproduct of particulate silica gel having an average particle diameter offrom about 3 to about 40 microns and an average pore size of from about50 to about 1000 Angstrom units with polyethyleniminopropyl trimethoxysilane having an average molecular weight of from about 400 to about1800.
 3. A method of obtaining essentially purified monoclonal antibodytype IgM from a sample of particulate-free mouse ascites fluidcontaining said monoclonal antibody which comprises:a. equilibratingsaid sample of particulate-free mouse ascites fluid to an ionic strengthequal to or less than the ionic strength of the lower ionic strengthbuffer used for gradient elution in the subsequent chromatographicseparation and recovery step and to a pH greater than the pI of themonoclonal antibody; and b. separating and recovering said monoclonalantibody type IgM from said sample by employing liquid chromatographicmeans wherein the chromatographic packing consists essentially of thereaction product of particulate silica gel having an average particlediameter of from about 3 to about 40 microns and an average pore size offrom about 50 to about 1000 Angstrom units with polyethyleniminopropyltrimethoxy silane having an average molecular weight of from about 400to about
 1800. 4. A method of obtaining essentially homongeneousmonoclonal antibody type IgM from a sample of mouse ascites fluidcontaining said monoclonal antibody which comprises separating andrecovering said monoclonal antibody from said sample by employing liquidchromatographic means wherein the chromatographic packing consistsessentially of particulate silica gel having an average particlediameter of from about 3 to about 40 microns and an average pore size offrom about 50 to about 1000 Angstrom units to which polyethyleniminefunctions are bound in adsorbed crosslinked form.
 5. A method ofobtaining essentially purified monoclonal antibody type IgM from asample of particulate-free mouse ascites fluid containing saidmonoclonal antibody which comprises:a. equilibrating said sample ofparticulate-free mouse ascites fluid to an ionic strength equal to orless than the ionic strength of the lower ionic strength buffer used forgradient elution in the subsequent chromatographic separation andrecovery step and to a pH greater than the pI of the monoclonalantibody; and b. separating and recovering said monoclonal antibody typeIgM from said sample by employing liquid chromatographic means whereinthe chromatographic packing consists essentially of particulate silicagel having an average particle diameter of from about 3 to about 40microns and an average pore size of from about 50 to about 1000 Angstromunits to which polyethylenimine functions are bound in adsorbedcrosslinked form.